Modulating cellular senescence by amphiphysin-1

ABSTRACT

The present invention relates to nucleic acid sequences and proteins involved in senescence and particularly, to nucleic acid sequences and proteins including amphiphysin and caveolin involved in cellular senescence and their use.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to nucleic acid sequences and proteinsinvolved in senescence and particularly, to nucleic acid sequences andproteins involved in cellular senescence and their use.

2. Description of the Related Art

The mechanism on senescence (also, called “aging”) was intensivelystudied and a variety of hypotheses were suggested. The hypothesiscomprises (a) free radical theory of aging (Harman D, Proc. Natl. Acad.Sci., 78, 7124-7128(1981)), (b) crosslinking theory of aging (BjorkstenJ., J. Am. Geriatr Soc., 16, 408-423(1968)), (c) mitochondrial theory ofaging (Lee C M et al., Free Radic. Biol. Med., 22, 1259-1269(1997); andWallace D C et al., Biofactors, 7, 187-190(1998)), and (d) geneticprogram theory of aging (Harley C B et al., Curr. Opin. Genet. Dev., 5,249-255(1995)).

Moreover, the senescence has been investigated in cellular level, i.e.,cellular senescence. According to the investigation, senescent cell ischaracterized by (a) arrest of cell cycle at G1 phase, (b) diminishedphysiological functions (Goldstein, Science, 249:1129-1133(1990);Campisi J., Cell, 84:497-500(1996)), and (c) resistance toapoptotic-programmed-cell death (Wang E., Cancer Res.,55:2284-2292(1995)).

A large variety of studies on cellular senescence have been made withhuman fibroblasts since the cells are considered to reflect a senescencephenomenon in individual level (Campisi J., Cell, 84:497-500(1996)).

Meanwhile, the patent applications related to nucleic acid and proteinsassociated with aging process, disclosed in WO 99/52929 and WO 01/23615.

As described above, a variety of theories have been proposed, thereremains a need of more evident elucidation for cellular senescence, aneed of specific biomarker for identifying senescent cell, and a need ofbiomolecule for modulating cellular senescence.

In particular, the prospect of reversing senescence and restoring normalphysiological function has an importance in certain diseases associatedwith senescence, for example, Werner Syndrome and Hutchinson-GilfordSyndrome.

Throughout this application, various patents and publications arereferenced and citations are provided in parentheses. The disclosure ofthese patents and publications in their entities are hereby incorporatedby references into this application in order to more fully describe thisinvention and the state of the art to which this invention pertains.

SUMMARY OF THE INVENTION

In one aspect of this invention, there is provided a method fordetecting a senescent cell comprising determining the amount of aprotein involved in cellular senescence of a cell, wherein the proteinis one or more selected from the group consisting of amphiphysin proteinand caveolin protein.

In another aspect of this invention, there is provided a method fordetecting a senescent cell comprising determining the amount of apolynucleotide encoding a protein involved in cellular senescence of acell, wherein the protein is one or more selected from the groupconsisting of amphiphysin protein and caveolin protein.

In still another aspect of this invention, there is provided acomposition for modulating cellular senescence comprising the effectiveamount of a protein involved in cellular senescence, wherein the proteinis selected from the group consisting of amphiphysin protein andcaveolin protein.

In further aspect of this invention, there is provided a composition formodulating cellular senescence comprising the effective amount of apolynucleotide encoding a protein involved in cellular senescence,wherein the protein is selected from the group consisting of amphiphysinprotein and caveolin protein.

In still further aspect of this invention, there is provided acomposition for modulating cellular senescence comprising the effectiveamount of an antisense oligonucleotide which hybridizes to apolynucleotide encoding a protein involved in cellular senescence andthereby inhibits the polynucleotide from expressing the protein, whereinthe protein is selected from the group consisting of amphiphysin proteinand caveolin protein.

In another aspect of this invention, there is provided a composition formodulating cellular senescence comprising the effective amount of amethylating agent or a demethylating agent, in which the agentmethylates or demethylates bases of a polynucleotide encoding caveolinprotein.

In still another aspect of this invention, there is provided acomposition for modulating cellular senescence comprising the effectiveamount of dominant negative amphiphysin-1 gene.

In further aspect of this invention, there is provided a method formodulating cellular senescence in a patient in need thereof, comprisingadministering to the patient the effective amount of a protein involvedin cellular senescence, wherein the protein is selected from the groupconsisting of amphiphysin protein and caveolin protein.

In still further aspect of this invention, there is provided a methodfor modulating cellular senescence in a patient in need thereof,comprising administering to the patient the effective amount of apolynucleotide encoding a protein involved in cellular senescence,wherein the protein is selected from the group consisting of amphiphysinprotein and caveolin protein.

In another aspect of this invention, there is provided a method formodulating cellular senescence in a patient in need thereof, comprisingadministering to the patient the effective amount of an antisenseoligonucleotide which hybridizes to a polynucleotide encoding a proteininvolved in cellular senescence and thereby inhibits the polynucleotidefrom expressing the protein, wherein the protein is selected from thegroup consisting of amphiphysin protein and caveolin protein.

In still another aspect of this invention, there is provided a methodfor modulating cellular senescence in a patient in need thereof,comprising administering to the patient the effective amount of amethylating agent or a demethylating agent, in which the agentmethylates or demethylates bases of a polynucleotide encoding caveolinprotein.

In further aspect of this invention, there is provided a method foridentifying a substance affecting the senescence of a cell, whichcomprises: (a) culturing the cell in the presence of the substance to betested; (b) isolating a protein from the cell; (c) contacting theisolated protein with an antibody specific to a protein involved incellular senescence, wherein the protein is selected from the groupconsisting of amphiphysin protein and caveolin protein; and (d)determining the amount of the isolated protein bound to the antibody.

In still further aspect of this invention, there is provided a methodfor identifying a substance affecting the senescence of a cell, whichcomprises: (a) culturing the cell in the presence of the substance to betested; (b) isolating RNA from the cell; (c) contacting the isolated RNAwith a polynucleotide encoding a protein involved in cellularsenescence, wherein the protein is selected from the group consisting ofamphiphysin protein and caveolin protein; and (d) determining the amountof the isolated RNA hybridized to the polynucleotide encoding a proteininvolved in endocytosis.

In another aspect of this invention, there is provided a kit fordetecting a senescent cell comprising a probe derived from apolynucleotide encoding a protein involved in cellular senescence,wherein the protein is selected from the group consisting of amphiphysinprotein and caveolin protein.

In still another aspect of this invention, there is provided a biomarkerfor identifying cellular senescence comprising a protein involved incellular senescence, wherein the protein is selected from the groupconsisting of amphiphysin protein and caveolin protein.

In further aspect of this invention, there is provided a biomarker foridentifying cellular senescence comprising a polynucleotide encoding aprotein involved in cellular senescence, wherein the protein is selectedfrom the group consisting of amphiphysin protein and caveolin protein.

Accordingly, it is an object of this invention to provide a method fordetecting a senescent cell.

It is another object of this invention to provide a composition formodulating cellular senescence.

It is still another object of this invention to provide a method formodulating cellular senescence in a patient in need thereof.

It is further object of this invention to provide a method foridentifying a substance affecting the senescence of a cell.

It is still further object of this invention to provide a kit fordetecting a senescent cell.

It is another object of this invention to provide a biomarker foridentifying cellular senescence.

Other objects and advantages of the present invention will becomeapparent from the detailed description to follow taken in conjugationwith the appended claims and drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a confocal microphotograph representing reduced endocytosis insenescent cell;

FIG. 2 is a confocal microphotograph representing the internalization oftrasferrin with increase of trasferrin treatment time in old and youngcells;

FIG. 3 is a confocal microphotograph showing pulse-chasing transferrinuptake;

FIG. 4 is a confocal microphotograph showing the internalization oftransferrin in induced senescent cell by H₂O₂ or hydroxyurea;

FIG. 5 is a photograph showing the results of western blotting foranalyzing the expression of proteins associated with cellularsenescence;

FIG. 6 is a photograph showing the results of western blotting foranalyzing the expression of proteins associated with cellular senescencein induced senescent cell by H₂O₂ or hydroxyurea;

FIG. 7 is a photograph showing the results of northern blotting foranalyzing mRNA encoding proteins associated with cellular senescense;

FIG. 8 shows a genetic map of the vector carrying cDNA encoding humanamphiphysin-1 used in Example VII;

FIG. 9 shows a genetic map of the expression vector carrying cDNAencoding human amphiphysin-1 constructed in Example VII;

FIG. 10 shows a photograph representing image observed with fluorescencemicroscope for analyzing amphiphysin-1 expression in microinjectedcells;

FIG. 11 represents a genetic map of the expression vector carryingdominant negative amphiphysin-1 gene;

FIG. 12 represents a confocal microphotograph demonstrating thesuppressed endocytic function of young cells treated with dominantnegative amphiphysin-1 gene;

FIG. 13 a shows a photograph representing the results of westernblotting for analyzing the activation (phosphorylation) of Erk-1/2kinase in young and middle cells;

FIG. 13 b shows a photograph representing the results of westernblotting for analyzing the activation (phosphorylation) of Erk-1/2kinase in old cells;

FIG. 14 shows a photograph representing the results of western blottingfor analyzing the expression of caveolin subtypes, that is, caveolin-1,caveolin-2 and caveolin-3, in young, middle and old cells;

FIG. 15 shows a photograph representing the results ofimmunoprecipitation, which verifies the interaction between epidermalgrowth factor receptor (EGFR) and caveolin-1 in young and old cells;

FIG. 16 is an electron microphotograph showing caveolae structure inyoung and old cells;

FIG. 17 represents a genetic map of the expression vector carryingcaveolin-1 cDNA constructed in Example XV;

FIG. 18 represents a photograph showing the results of western blottingfor young cells transformed with caveolin-1 cDNA;

FIG. 19 represents a confocal microphotograph indicating that caveolin-1expression is dramatically decreased by antisense oligonucleotide;

FIG. 20 is a photograph showing the results of western blotting foranalyzing Erk-1/2 activation upon transfection of antisenseoligonucleotie to caveolin-1;

FIG. 21 is a confocal microphotograph representing activation andlocalization of Erk-1/2 upon epidermal growth factor (EGF) stimulationin young and old cells;

FIG. 22 shows a confocal microphotograph representing activation andlocalization of Erk-1/2 upon EGF stimulation, in young and old cells,after downregulation of caveolin-1, that is after transfection ofantisense oligonucleotide to caveolin-1;

FIG. 23 a is a photograph representing the results ofsenescence-associated β-galactosidase activity staining for young cellstreated with demethylating agent, 5-aza-deoxycytidine; and

FIG. 23 b shows a photograph representing the results of westernblotting for young cells treated with demethylating agent,5-aza-deoxycytidine.

DETAILED DESCRIPTION OF THIS INVENTION

The present invention, in principle, is directed to nucleic acids andproteins modulating cellular senescence. The inventors have found thatamphiphysin and caveolin are responsible for cell senescence in eachdifferent manner, as demonstrated in Example.

The process of receptor-mediated endocytosis via clathrin-coated vesicleis composed of several steps, which include recruitment of the clathrincoats and fission of the coated bud (Schmid, S. L. Annu. Rev. Biochem.,66:511-548(1997)). After binding of ligand to the receptor, such asepidermal growth factor (hereinafter referred at as “EGF”), receptortyrosine kinase phophorylates clathrin, which in turn can provide abinding site for Src-homology-3 (SH3) domain of amphiphysin (Slepnev, V.I. et al., Science, 281:821-824(1998); Wang, L. H. et al., J. Biol.Chem., 270:10079-10083(1995); and Ramjaun, A. R. et al., J. Neurochem.,70:2369-2376(1998)). Although its precise mechanism of action is notclear, amphiphysin-1 is thought to involve the recruitment andoligomerization at the neck of endocytotic buds (Schmid, S. L. Annu.Rev. Biochem., 66:511-548(1997); and Takei, K. et al., Nat. Cell Biol.,133-139(1999)). Amphiphysin-1 bridges the AP2/clathrin coat anddynamin-1 to make an endosomal vesicle (Slepnev, V. I. et al., Science,281:821-824(1998); Shupliakov, O., et al., Science, 276:259-263(1997);McMahon, H. T. et al., FEBS Lett., 413:319-322(1997); -David, C., etal., Proc. Natl. Acad. Sci., 93:331-335(1996); and Urrutia, R., et al.,Proc. Natl. Acad. Sci., 94:377-384(1997)). The carboxyl-terminal domainof amphiphysin recruits GTPase dynamin to pinch off the coated buds(David, C., et al., Proc. Natl. Acad. Sci., 93:331-335(1996); andUrrutia, R., et al., Proc. Natl. Acad. Sci., 94:377-384(1997).Disruption of the interaction of amphiphysin with either dynamin orclathrin and AP-2 inhibits clathrin-mediated endocytosis (Slepnev, V.1.et al., Science, 281:821-824(1998); Shupliakov, O., et al., Science,276:259-263(1997); and Wigge, P. et al., Curr. Biol., 7:554-560(1997)).These findings indicate that amphiphysin may act as a regulated linerprotein that couples clathrin-mediated budding of endocytotic vesiclesto dynamin-mediated vesicle fission. Furthermore, it have been reportedthat amphiphysin has several subtypes and amphiphysin-2 also has a SH3domain and has a binding-affinity to dynamin as amphiphysin-1.

Caveolae are vesicular invaginations of the plasma membrane with adiameter of 50-100 nm and are involved in endocytosis such astranscytosis and ptocytosis and signal transduction (Engelman, J. A. etal., FEBS Lett., 428:205(1998)). Caveolin, a 21-24 kDa integral membraneprotein, is a principal structural component of caveolae membranes invivo. The stable expression of caveolin-1 or -3 gene to the mammaliancells without caveolin induced the formation of caveolae structures(Lipardi, C. et al., J. Cell Biol., 140:617(1998)). Caveolin has beenfound as several subtypes in vivo. Caveolin-1 is a key constituent ofcaveolae structures. Caveolin-2, is expressed ubiquitously in most celltypes, supposedly forming a hetero-oligomer in basolaterally localizedcaveolae (Scheiffele, P. et al., J. Cell Biol., 140:795(1998)). It hasbeen reported that the expression of caveolin-3 is restricted tostriated muscle cells (Tang, Z. et al., J. Biol. Chem., 271:2255(1996)).

I. Method for Detecting a Senescent Cell and Method for Identifying aSubstance Affecting Cellular Senescence

The present methods employ proteins involved in endocytosis such asamphiphysin protein and caveolin protein.

In the present method, the signal indicating cell senescence is detectedeither by measuring the decreased level of, amphiphysin protein in cellor by measuring the increased level of caveolin protein in cell.

The term “senescence” is used herein to have the same meaning as“aging.” The term “old cell” is used herein to have the same meaning as“senescent cell.” Unless otherwise defined, all technical and scientificterms used herein have the same meaning as commonly understood by one ofordinary skill in the art to which this invention belongs. For example,the terms used herein may be found in Benjamin Lewin, Genes VII,published by Oxford University Press(2000); and Kendrew et al., TheEncyclopedia of Molecular Biology, published by Blackwell Science Ltd.(1994).

According to the preferred embodiment, the cell is derived frommammalian cell such as human cell.

Amphiphysin used in this invention may be selected from amphiphysinsubtypes as described above. It is preferred that the amphiphysinprotein is amphyamphiphysin-1, which has been known to a main subtype asmentioned previously. Furthermore, the caveolin protein used may beselected from caveolin-1, caveolin-2 and caveolin-3. It is preferredthat the caveolin used is caveolin-1 protein, which has also been knownto a main subtype as described previously.

In the present method which uses antibody against amphiphysin orcaveolin, the antibody may be obtained as methods known to those skilledin the art (Antibodies: A Laboratory Manual, Cold Spring HarborLaboratory (1988)). The antibody may be polyclonal or monoclonalantibody. Monoclonal antibody may be readily prepared through use ofwell-known techniques, such as those exemplified in U.S. Pat. No.4,196,265. The step of determining the amount of the isolated proteinbound to the antibody may be performed according to methods known tothose skilled in the art such as radioimmunoassay and enzyme-linkedimmunosorbent assay. These methods are generally based on the detectionof a label or marker such as radioactive, fluorescent, biological orenzymatic tags or labels.

In a preferred embodiment of this method, the method is conducted bywestern blotting method. The general procedure of western blottingmethod is disclosed in Peter B. Kaufma et al., Molecular and CellularMethods in Biology and Medicine, 108-121, CRC Press. The westernblotting for this invention, preferably, comprises the steps of (a)lysing a cell sample to be measured; (b) preparing a protein from thelysed cell; (c) denaturating the prepared protein in solution containingSDS and 2-mercaptoethanol; (d) performing SDS-polyacrylamide gelelectrophoresis; (e) transferrin the protein on gel to nitrocellulose(hereinafter referred to as “NC”) membrane; (f) reacting the protein onNC membrane with a primary antibody to amphiphysin or caveolin,advantageously, amphiphysin-1 or caveolin-1; (g) reacting the primaryantibody with a secondary antibody conjugated to enzyme catalyzingcalorimetric reaction; (h) inducing the calorimetric reaction by addinga substrate for the enzyme of (g); and (i) measuring the intensity ofcolor developed by the enzyme (g).

Preferred enzyme for colorimetric reaction includes, but not limited to,alkaline phosphatase, 1-galactosidase, and horse radish peroxidase.Where using alkaline phosphatase, bromochloroindolylphosphate (BCIP),nitro blue tetrazolium (NBT) and ECF may be used as a substrate; in thecase of using horse radish peroxidase, chloronaphtol,aminoethylcarbazol, diaminobenzidine and luminol may be used as asubstrate.

As described in Examples, the inventors have found that the level ofamphiphysin in cell is dramatically decreased in senescent stage andvice versa for caveolin. Therefore, the present method may be carriedout qualitatively. For example, the strength and the thickness ofwestern blotting band for detecting amphiphysin may be found to bedramatically decreased to the extent capable of detecting visually; inthe case of western blotting for detecting caveolin, the strength andthe thickness of the resulting band may be found to be dramaticallyincreased. Consequently, with comparing the western blotting bandderived from senescent cell to one derived from young cell, thesenescence can be easily detected.

Moreover, the present method may be carried out in a quantitativemanner. For example, the bands resulted from western blotting may betransformed to quantitative data with densitometor. In a specificexample for analyzing 40′ μg protein, if the level of expression ofamphiphysin-1 in tested cell is 45 times less than young cell, thetested cell may be considered senescent.

The present methods employ a polynucleotide coding for proteins involvedin endocytosis such as amphiphysin protein and caveolin protein.

In this method, the signal indicating cell senescence is detected eitherby measuring the decreased level of a polynucleotide encodingamphiphysin protein in cell or by measuring the increased level of apolynucleotide encoding caveolin protein in cell.

According to the preferred embodiment, the cell is derived frommammalian cell such as human cell. It is preferred that thepolynucleotide is one conding for amphiphysin-1 or caveolin-1 protein.It is preferred that the polynucletide used in this method is gDNA(genomic DNA), cDNA and mRNA.

In a preferred embodiment of this method, the method may be conducted bynorthern blotting method. The general procedure of northern blottingmethod is disclosed in Peter B. Kaufma et al., Molecular and CellularMethods in Biology and Medicine, 102-108, CRC Press. The northernblotting for this invention, preferably, comprises the steps of (a)preparing RNA from cell to be tested; (b) performing electrophoresiswith the prepared RNA; (c) transferrin the RNA to nylon or NC membrane;(f) hybridizing the transferred RNA with radio-labeled oligonucleotideprobe complementary to amphiphysin mRNA or caveolin mRNA,advantageously, amphiphysin-1 or caveolin-1; and (g) measuring theintensity of the resulting band.

As described in Examples, the inventors have revealed that the level ofamphiphysin RNA in cell is dramatically decreased in senescent stage andvice versa for caveolin. Therefore, the present method may be carriedout qualitatively. For example, the strength and the thickness ofnorthern blotting band for detecting amphiphysin RNA may be found to bedramatically decreased to the extent capable of detecting visually; inthe case of northern blotting for detecting caveolin RNA, the strengthand the thickness of the resulting band may be found to be dramaticallyincreased. Consequently, with comparing the northern blotting bandderived from senescent cell to one derived from young cell, thesenescence can be conveniently detected.

Moreover, the present method may be carried out in a quantitativemanner. For example, the bands resulted from northern blotting may betransformed to quantitative data with densitometor. In a specificexample for analyzing 50 μg of amphiphysin-1 RNA, if the strength ofband for tested cell is 15 times less than young cell, the tested cellmay be considered senescent.

II. Composition for Modulating Cellular Senescence

The composition for modulating cellular senescence of this inventioncomprises biomolecule capable of modulating cellular senescence. Thebiomolecule includes: (a) protein such as amphiphysin protein andcaveolin protein; and (b) a polynucleotide such as one encodingamphiphysin protein or caveolin protein. In addition, the biomoleculeincludes antisense oligonucleotide capable of hybridizing to apolynucleotide encoding amphiphysin protein or caveolin protein.Meanwhile, the composition of this invention comprises a methylatingagent or a demethylating agent to methylate or demethylate bases of apolynucleotide encoding caveolin protein.

As described above, while it is conceivable that the proteins may bedelivered directly, a preferred embodiment involves providing apolynucleotide encoding amphiphysin or caveolin.

According to the preferred embodiment, the cell is derived frommammalian cell such as human cell. It is preferred that the proteininvolve in endocytosis is amphiphysin-1 or caveolin-1 protein.

In the composition containing a polynucleotide, it is preferred that thepolynucletide is gDNA or cDNA and is carried by expression vector foreucaryotic cell. The polynucleotide encoding amphiphysin-1 includes,preferably, nucleotide sequence coding for amino acids sequencerepresented by SEQ ID NO:2 and, more preferably, nucleotide sequencecorresponding to nucleotides 111-2195 of nucleotide sequence representedby SEQ ID NO:1. The polynucleotide encoding caveolin-1 includes,preferably, nucleotide sequence coding for amino acids sequencerepresenteds by SEQ ID NO:4 and, more preferably, nucleotide sequencecorresponding to nucleotides 26-559 of nucleotide sequence representedby SEQ ID NO:3.

The expression vector used in this invention expresses foreign gene ineucaryotic host, preferably mammalian cell, more preferably-human cell.The promoter in the expression vector may be derived from the genome ofmammalian cells (e.g., metallothionein promoter) or from mammalianviruses (e.g., adenovirus late promoter; vaccinia virus 7.5K promoter).Further, it is also possible, and may be desirable, to utilize promoteror control sequences normally associated with the desired gene sequence,provided such control sequences are compatible with the host cellsystems. A number of viral based expression systems may be utilized, forexample, commonly used promoters are derived from polyoma, Adenovirus 2,and most frequently Simian Virus 40 (SV40). It is desired to incorporateinto the transcriptional unit an appropriate polyadenylation site. Theexample of commercial vectors used for this invention includes pcDNA 3(Invitrogen; containing cytomegalo virus promoter and polyadenylationsignal), pSI (Promega; containing SV 40 promoter and polyadenylationsignal), pCI (Promega; containing containing cytomegalo virus promoterand polyadenylation signal), and pREP7 (Invitrogen; RSV promoter and SV40 polyadenylation signal).

Furthermore, in the composition containing a polynucleotide encodingamphiphysin or caveolin, the polynucleotide may be delivered using viralvectors designed for gene therapy. For example, the delivery systemsincludes, but not limited to, (a) adenoviral vectors(Stratford-Perricaudet and Perricaudet, In: Human Gene Transfer, Eds.,Cohen-Haguenauer and Boiron, Editions John Libbey Eurotest, France,51-61(1991); and Stratford-Perricaudet et al., Hum. Gene Ther.,1:241-256(1991)); (b) adeno-associated virus vectors (LaFace et al.,Virology, 162:483-486(1998); Zhou et al., Exp. Hematol.,21:928-933(1993); and Walsh et al., J. Clin. Invest.,94:1440-1448(1994)); and (c) retroviral vectors such as engineeredvariant of the Moloney murine leukemia virus (Kasahara et al., Science,266:1373-1376(1994)).

In the present composition containing antisense oligonucleotide, theantisense oligonucleotide may hybridize, under intracellular conditions,to target DNA or RNA. Targeting double-stranded DNA with an antisenseoligonucleotide leads to triple-helix formation; targeting RNA leads todouble-helix formation. Antisense oligonucleotide may be designed tobind to the promoter and other control regions, exons and introns oftarget gene. The antisense oligonucleotide used in this invention may besubstantially complementary to target polynucleotide. That is, theantisense constuct may have some base mismatches to target gene. It ismore preferred that the antisense oligonucleotide hybridizes to apolynucleotied encoding caveolin protein, advantageously, caveolin-1protein. It is the most preferred that the antisense oligonucleotidehybridizes to translational initiation region of caveolin-1 mRNA.

In the present invention containing a methylating agent or ademethylating agent, the agent regulates the level of methylation forbases of caveolin gene. As described in Example, caveolin gene with lessmethylation level exhibits greater expression level. In a preferredembodiment, the caveolin gene to be methlyated or demethylated iscaveolin-1 gene. More preferably, the modified region is a promoter ofcaveolin-1 gene and the most preferably, CpG island from the promoter ofcaveolin-1 gene. Example of methylating agent used in this inventionincludes, but not limited to, methylazoxymethanol acetate, Temozolomideand N-methyl-N-nitrosourea. Non-limiting example of demethylating agentused in this invention includes 5-aza-deoxycytidine, 5-azacytidine,6-azacytidine and 8-azaguanine.

As a composition for modulating cellular senescence, the presentinvention provides a composition comprising the effective amount ofdominant negative amphiphysin-1 gene. The dominant negativeamphiphysin-1 gene, which has been known to block the function ofamphiphysin-1 (Shupliakov, O. et al., Science, 276:259(1997); and WiggeP., Curr. Biol., 7:554(1997)). The treatment with dominant negativeamphiphysin-1 gene leads to cellular senescence as described in Example.According to a preferred embodiment, the dominant negative amphiphysin-1gene is a polynucleotide encoding a polypeptide comprising the aminoacid sequence 250 to 588 represented by SEQ ID NO:2.

III. Method for Modulating Cellular Senescence

In the method of this invention, the effective amount of biomoleculerelated to amphiphysin or caveolin is typically administered to a cell.In particular, a polynucleotide encoding amphiphysin or caveolin orantisense oligonucleotide thereof may be introduced in vivo or ex vivoin accordance with the following methods: (a) microinjection (Capecchi,M. R., Cell, 22:479(1980)); (b) calcium phosphate co-precipitation(Graham, F. L. et al., Virology, 52:456(1973)); (c) electroporation(Neumann, E. et al., EMBO J., 1:841(1982)); (d) liposome-mediatedtransfection (Wong, T. K. et al., Gene, 10:87(1980)); (e) DEAE-dextrantreatment (Gopal, Mol. Cell Biol., 5:1188-1190(1985)); and (f) particlebombardment (Yang et al., Proc. Natl. Acad. Sci., 87:9568-9572(1990)).

According to a preferred embodiment, the cell used is derived frommammalian cell, more preferred, human cell. Preferably, protein,polynucleotide and antisense oligonucleotide administered are related toamphiphysin-1 or caveolin-1. It is preferred that the polynucleotideadministerd is gDNA or cDNA. More preferably, the polynucleotideadministered is carried on expression vector for eucaryotic cell. In apreferred embodiment of a method using antisense construct, theantisense oligonucleotide is substantially complementary to the geneencoding caveolin, more preferably, caveolin-1. The most preferableembodiment comprises the antisense oligonucleotide hybridizes totranslational initiation region of caveolin-1 mRNA.

In the present method using a methylating agent or a demethylatingagent, the agent regulates the level of methylation for bases ofcaveolin gene. In a preferred embodiment, the caveolin gene to bemethlyated or demethylated is caveolin-1 gene. More preferably, themodified region is a promoter of caveolin-1 gene and the mostpreferably, CpG island from the promoter of caveolin-1 gene. Example ofmethylating agent used in this invention includes, but not limited to,methylazoxymethanol acetate, Temozolomide and N-methyl-N-nitrosourea.Non-limiting example of demethylating agent used in this inventionincludes 5-aza-deoxycytidine, 5-azacdytidine, 6-azacytidine and8-azaguanine.

The common descriptions of between I, II and III are abbreviated inorder to avoid the complexity of this specification leading to unduemultiplicity

IV. Kits and Biomarkers

As indicated above, the present invention provides a kit for detecting asenescent cell. All the essential materials and reagents required fordetecting a senescent cell may be assembled together in a kit. The probeused may be useful for hybridization to DNA or RNA isolated from a cellto be tested. Furthermore, the probe used may be primer primer for usein any molecular biology assay known to those of skill in the art suchas PCR and RT-PCR. Also included may be enzymes suitable foramplification nucleic acids such as Taq polymerase, dNTP mixture andbuffers to provide the necessary reaction mixture for amplification.

In a preferred embodiment, the probe is derived from the polynucleotideencoding amphiphysin-1 protein or caveolin-1 protein. The probe,preferably, is immobilized on a solid support. Solid supports suitablefor use in the kit of this invention are known to those of skill of theart, which includes glasses, plastics, polymers, metals, metalloids,ceramics and organics. According to more preferred embodiment, thisinvention provides a kit comprising an array of probes derived from thepolynucleotide encoding amphiphysin-1 protein or caveolin-1 protein. Thegeneral techniques for microarray containing solid supports have beendisclosed in many publications such as WO 89/10977, U.S. Pat. Nos.5,202,231, 5,002,867 and 5,143,854.

According to preferred embodiment of this invention, the kit furthercomprises a label for detecting the presence of the probe. The labelallows detection of hybridization of between the probe and nucleotidesisolated from sample to be tested. The most common label is radioactivematerial such as ³H, ¹⁴C and ³²P.

The present invention provides a biomarker for identifying cellularsenescence. In preferred embodiments, the protein or the polynucleotiesuitable for this invention, is derived from amphiphysin-1 orcaveolin-1.

The following specific examples are intended to be illustrative of theinvention and should not be construed as limiting the scope of theinvention as defined by appended claims.

EXAMPLE I Cell Culture

I-1: Culture for Human Foreskin Fibroblast

Foreskin fibroblast was isolated and cultured according to the methodprovided by Boyce and Ham (Boyce S T. and Ham R G., J. Invest. Dermato.,81:33-40(1983)) as follows: First, foreskin was obtained from 7-year-oldKorean male and was stripped to give pieces, after which the foreskinpieces was added to 10 ml of Hank's salt solution (Gibco BRL) containing0.25% collagenase. Following incubating for 90 min. at 37° C. inCO₂-controlled (5%) incubator, epithelium and dermis were separated fromeach other. To the separated dermis, 1 ml of tryp sin solution (0.25%)was added and the resulting solution was added to 10 ml of DMEM(Dulbecco's modified Eagle medium: Sigma) containing 100 μg/ml ofstreptomycin and 100 units/ml of penicillin, followed by incubating for10 min. at 37° C. The yielded foreskin fibroblast were washed with 10 mlof PBS and in DMEM (supplemented with 10% FBS and antibiotics) wereserially passaged as follows: The incubator was maintained to theatmosphere of 5% CO₂ and the temperature of 37° C., DMEM was renewedonce per 3 days and subconfluency (about 80-90%) was kept and subculturewas performed at a 1:4 ratio. The cells, cultured with less than 25population doublings, were considered presenescent cells (or youngcells), which are highly proliferative, while cells with over 60population doublings were defined as senescent cells, which showeddelayed population doublings times (over 3 weeks).

I-2: Culture for Fetal Lung Fibroblast

Fetal lung fibroblast, IMR-90, was purchased from ATCC(CCL-186). Theculture for IMR-90 was carried out in the same manner as the above.

EXAMPLE II Induction of Cellular Senescence

II-1: Induction of Cellular senescence with H₂O₂

The fibroblasts subcultured in Exmaple I-1, PDL of which are 16, wereplaced in culture plate and were kept to arrest cell cycle to G1 phasein incubator (37° C., 5% CO₂ and humidified) for a week. The cell cyclearrest was confirmed as follows: Following the fixation of the cellswith cold ethanol, the cells were stained for 30 min. at roomtemperature using PI staining solution (containing 50 μg/ml of Rnase Aand 50 μg/ml of propinium iodide in PBS). Thereafter, using FACS(fluorescence-activated cell sorter), the cell cycle was confirmed byobserving DNA phenotype, i.e., 2n or 4n. The cells in G1 phase wereshowed 2n of DNA phenotype.

The fibroblasts arrested in G1 phase were treated with 400 μM H₂O₂ andthen incubated for 3 hrs., followed by washing with 10 ml of PBS. Then,the cells were subcultured at a 1:4 ratio and under normal conditionsfor cell culure (37° C., 5% CO₂ and humidified), cell culture wascontinuously performed. Following 7 days after the treatment, the cellswere determined in terms of senescence using senescence-associated3-galactosidase activity staining as described in Example III.

II-2: Induction of Cellular senescence with Hydroxyurea

The fibroblasts subcultured in Exmaple I-1, PDL of which are 16, wereplaced in culture plate containing DMEM and were cultured in incubator(37° C., 5% CO₂ and humidified) for 14 hrs. Thereafter, the fibroblastswere treated with 400 μM Hydroxyurea and then incubated continuously.The medium was renewed once per 3 days with the addition of fresh 400 μMHydroxyurea. Following 14 days after the treatment, the cells weredetermined in terms of senescence using senescence-associated1-galactosidase activity staining as described in Example III.

EXAMPLE III Senescence-Associated β-Galactosidase Activity Staining

A senescence-associated J-galactosidase activity staining (hereinafterreferred to as “SA β-gal activity staining) was performed according tothe method of Dimri et al. (Dimri G P et al., Proc. Natl. Acad. Sci.,92:9363(1995)): The semiconfluent fibroblasts were washed twice with 10ml of PBS and fixed with 2% paraformaldehyde in PBS for 5 min. at roomtemperature. After washing with PBS, cells were incubated with SA β-galactivity staining solution (1 mg/ml of β-gal, 40 mM citric acid/sodiumphosphate buffer, pH 6.0, 5 mM potassium ferrocyanide/ferricyanide, 150mM NaCl and 2 mM MgCl₂) at 37° C. for 4 hrs. Young and old humanfibroblasts were observed with phase contrast microscopy. As a result ofthe observation, human fibroblast showed β-galactosidase activity fromPDL 50, IMR 90 from PDL 65, cells treated with H₂O₂ from 10 days aftertreatment and cells treated with hydroxyurea from 14-20 days aftertreatment, which demonstrate the entry of cellular senescence.

EXAMPLE IV Evaluation of Alteration of Endocytosis

IV-1; Observation of Reduced Endocytosis in Senescent Cell

To investigate the functional changes of receptor-mediated endocytosisin senescent cell, the internalization of transferrin was observed. Thefibroblasts were plated onto cover glasses and incubated in incubator(37° C., 5% CO₂ and humidified), followed by the treatment of 25 μg/mltetramethylrhodamine-conjugated human transferrin (Molecular Probes) for5 min. After washing 10 ml of PBS, the cells were fixed with 4%paraformaldehyde in PBS for 10 min at room temperature, and then, nucleiof cells were stained with DAPI (Sigma Aldrich). Internalization offluorescent transferrin was monitored with confocal microscopy (Biorad,#MRC1024), which is shown in FIG. 1. In FIG. 1, panel A represents young(PDL 20) fibroblasts and senescent fibroblasts (PDL 54) and panel Brepresents IMR cells (PDL 32 and PDL 68, respectively). As demonstratedin FIG. 1, young fibroblasts and IMR cells took up fluorescenttransferrin readily and internalized transferrin was observed as typicalpunctuated crescent shapes in the perinuclear area. In contrast,senescent cells did not uptake transferrin as efficiently aspresenescent cells.

IV-2: Observation of Internalization of Transferrin with Increase ofTreatment Time

The fibroblasts were treated with 25 μg/mltetramethylrhodamine-conjugated human transferrin for 10, 20, 40 or 60min as described previously; and the experimental results are shown inFIG. 2. In FIG. 2, panels Y, M and O. represent young fibroblasts (PDL24), middle fibroblasts (PDL 38) and senescent fibroblasts (PDL 54),respectively. As shown in FIG. 2, cells with PDL 24 and PDL 38 took upfluorescent transferrin efficiently and internalized transferrin wasobserved in the perinuclear area in 10-min treatment, which wasincreased with the increase of treatment time, thereby giving greaterfluorescence intensity. In contrary to this, the senescent fibroblastsdid not uptake transferrin even after 60-min treatment.

IV-3: Pulse-Chasing of Transferrin Uptake

Twenty five μg/ml rhodamine-conjugated transferrin was pulsed on IMR 90cells for 5 min and then chased for 0, 5 and 10 min. After fixing asdescribed above, internalization of fluorescent transferrin wasmonitored with confocal microscopy (see FIG. 3). In FIG. 3, panels Y, Mand 0 represent young (PDL 26), middle (PDL 48) and senescent cells (PDL72), respectively. As shown in FIG. 3, young cells efficiently uptaketransferrin and localize it to perinuclear area just in 5 min and thenthe transferrin was quickly degraded after 10 min chasing. PDL 48 cellsrevealed a delayed and limited uptake of transferrin after 10 minchasing. PDL 72 cells nearly failed to uptake transferrin with the lapseof chasing time.

IV-4: Observation of Reduced Endocytosis in Artificially InducedSenescent Cell

The senescent cells which were artificially induced in Example II weretreated with 25 μg/ml rhodamine-conjugated transferrin as above andinternalization of fluorescent transferrin was monitored with confocalmicroscopy (see FIG. 4). As indicated in FIG. 4, not onlynaturally-occurring senescent cells through serial passage but alsoartificially-induced senescent cells by H₂O₂ or hydroxyurea (marked “+”)showed the reduced function of receptor-mediated endocytosis.

In summary, the inventors have revealed that senescent fibroblast cellsshow significantly reduced function of endocytosis and thus fail touptake a variety of ligands such as transferrin.

EXAMPLE V Analysis of Expression of Proteins Involved inReceptor-Mediated Endocytosis

To identify the molecular mechanism for such alteration in thereceptor-mediated endocytosis of senescent cells, the expression levelof several proteins involved in receptor-mediated endocytosis waschecked through western blotting experiment.

First, total cell lysates were extracted from subconfluent early, middleand late-passaged cells using lysis buffer (1% Triton X-100, 0.5% NP-40,50 mM Tris pH7.5, 150 mM NaCl, 1 mM EDTA, 1 mM PMSF, 5 #g/ml aprotinin,5 μg/ml leupeptin, 1 mM NaVO₄ and 1 mM NaF) and sonicated briefly, afterwhich the lysates were centrifuged at 14000×g for 10 min and thesupernatants were taken. With the supernatants, the proteinquantification was performed as Bradford method (Bradford, M., Anal.Biochem. 0.72:248-254(1976)) and 40 μg of protein equivalents wereboiled for 5 min in 5×SDS sample buffer (60 mM Tris-Cl, pH 6.8, 25%glycerol, 2% SDS, 14.4 mM 2-mercaptoethanol and 0.1% bromophenol blue).Cell lysates (10-15 μg of protein equivalents) were electrophoresd on 8%polyacrylamide gel using electrophoresis kit (Biorad) and thentransferred to nitrocellulose membranes using transfer kit (Biorad). Theblots were blocked with TTBS (Tris buffered saline with Tween 20)containing 5% non-fat dry milk (Difco) for 1 hr at room temperature.,The blots were immunoblotted with the respective primary antibody inTTBS with 5% non-fat dry milk for 1 hr. at room temperature, washedthree times with TTBS, and incubated with horseradishperoxidase-conjugated anti-mouse secondary antibody (Jackson ImmunoResearch Laboratory). In the primary antibodies, anti-dynamin antibody,anti-α-adaptin, anti-β-adaptin and anti-clathrin heavy chain antibodywere purchased from Transduction Laboratories, monoclonal antibody toamphiphysin-1 was prepared by Dr. Kim from Chungbuk National University(Jin, Y., Kim et al. (In press), Production and characterization ofmonoclonal antibodies against amphiphysin, Exp. Mol. Med.), andanti-phosphotyrosin antibody and anti-transferrin receptor antibody wereSanta Cruz Biotechnology. The signals were finally visualized by anenhanced chemiluminescence system (ECL kit, Amersham Pharmacia Biotech),which are found in FIG. 5.

As demonstrated in FIG. 5, only amphiphysin-1, but none of otherendocytotic protein tested, was significantly reduced in senescentcells. The unique reduction in expression of amphiphysin-1 protein wasobserved in the senescent cells of both foreskin fibroblasts and IMR 90cells. Moreover, the senescent cells which were induced by H₂O_(2 or)hydroxyurea (marked “+”) gave the same results as shown in FIG. 5 (seeFIG. 6).

These results indicate that the cellular senescence process isaccompanied with down regulation of amphiphysin-1.

EXAMPLE VI Northern Analysis of mRNA Encoding Proteins Involved inReceptor-Mediated Endocytosis

To investigate the exact mechanism for the reduced level ofamphiphysin-1 protein in senescent cells, which was analyses in ExampleV, northern blotting was carried out as follows:

The RNA was isolated from human fibroblasts of Example I using acidguanidinium thiocyanate-phenol-chloroform, mixed with formaldehydesample buffer (5×MOPS, 17.5% formaldehyde and 50% formamide), and thenelectrophoresed on 1% agarose gel using electrophoresis kit (Hoefer).Following the electrophoresis, the RNA was transferred to nitrocellulosemembrane and was cross-linked to the membrane using auto UV crosslinker(Stratagen). Then, the hybridization was performed with P³²-labelledprobe (comprising bases 111-1116 of amphiphysin-1 cDNA) and theresulting autoradiograms were obtained (see FIG. 7). In FIG. 7, panels.Y, M and 0 represent PDL 27, PDL 36 and PDL 60 fibroblasts,respectively. According to FIG. 7, it was elucidated that the level ofamphiphysin mRNA was reduced with a progression of senescence.

Theses results indicate that the cellular senescence process isaccompanied with down regulation of amphiphysin-1 at transcriptionallevel.

EXAMPLE VII Cloning of Amphiphysin-1 Gene

The cDNA encoding the full length of human amphiphysin-1 has anucleotide sequence represented by SEQ ID NO:1 and the vector carryingthe cDNA was obtained from Chungbuk National University (Korea). Thevector was constructed in such a manner that the cDNA was insertedbetween BamHI and EcoRI restriction sites of pGEX-2T vector (Pharmacia)and thereby amphiphysin-1 and glutathione-S-transferase were expressedin fused form. FIG. 8 shows genetic map of the final vector carryingcDNA encoding human amphiphysin-1. The full length cDNA was amplified byPCR using a set of primers: 5′-AACTGTCCACCATGGCCGACATCAAGACGGGC-3′ and5′-GGATCCCTAATCTAAGCGTCGGGT-3′. The PCR amplification was performed for30 cycles using Pyrobest Taq polymerase (Takara) in accordance withfollowing temperature sets: 55° C. for 30 sec (annealing), 72° C. for1.5 min (extension) and 92° C. for 30 sec (denaturation). The amplifiedcDNA was cloned into pT7 blue vector (Novagen) and its base sequence wasdetermined. After digestion with HindIII and BamHI, the cDNA wassubcloned into pcDNA3 vector (Invitrogen: containing promoter andpolyadenylation signal of cytomegalo virus) in order to microinject theamplified cDNA of amphiphysin-1 to fibroblast. The pcDNA3 containingcDNA of amphiphysin-1 was showed in the genetic map of FIG. 9.

EXAMPLE VIII Restoration of Endocytid Function in Senescent Cell byAmphiphysin-1 Gene

VIII-1: Microinjection of Amphiphysin-1 Gene

Senescent fibroblasts (PDL 58) in Example I were placed onto cover glassand incubated for 24 hrs. in DMEM with on FBS in incubator (37° C., 5%CO₂ and humidified). Then, 10⁻¹⁴ l of amphiphysin-1 gene cloned intopcDNA3 of Example VII (10 ng/ml) and 10⁻¹⁴ l of rabbit IgG (Sigma, 5mg/ml) were microinjected into nucleus of senescent fibroblast. Thevector was diluted to 10 ng/ml in the microinjection buffer (50 mMHEPES, pH 7.2, 100 mM KCl, 50 mM NaPO₄). The diluted vector wasmicroinjected into nucleus using transjector 5426 (Eppendorf) andmicromanipulator (Eppendorf).

VIII-2: Analysis of Amphiphysin-1 Expression in Microinjected Cell

Using double immunofluorescent staining method, the expression ofamphiphysin-1 in microinjected cell was analyzed. Following 24 hrs.incubation in DMEM without FBS after microinjection, the cells werefixed with 3.7% paraformaldehyde (in PBS) for 10 min. and thenpermeabilized with 0.3% Triton X-100 in PBS for 10 min at roomtemperature. The cells were sequentially incubated withanti-amphiphysin-1 antibody, FITC-conjugated anti-rat IgG antibody(Jackson Laboratory: 1:100 dilution) for 1 hr at 37° C., and thenrhodamine-conaugated anti-rabbit IgG antibody (Jackson Laboratory: 1:100dilution) for 1 hr at 37° C. The image was observed with fluorescencemicroscope (Zeiss, Axiovert25, CFL451210) (see FIG. 10). As shown inFIG. 10, anti-rabbit IgG gives red fluorescence emitted by rhodamine andamphiphysin-1 is determined by green fluorescence emitted by FITC ofsecondary antibody. The green fluorescence demonstrating the existenceof amphiphysin-1 was observed in cytoplasm.

Therefore, it is elucidated that amphiphysin-1 protein is expressed inthe microinjected cells in Example VIII-1.

VIII-3: Analysis of Restoration of Endocytic Function in Senescent Cell

Microinjected cells were incubated for 24 hrs. in DMEM without FBS inincubator (37° C., 5% CO₂ and humidified). Then, the cells were, treatedwith 125 ng/ml tetramethylrhodamine-conjugated transferrin for 30 min.,washed with 10 ml of PBS and fixed with 10 ml of 4% formaldehyde (inPBS) for 10 min. The immunofluorescence staining was performed asdescribed in Example VIII-2. By means of fluorescence microscope (Zeiss,Axiovert 100) microinjected cells and uptaken transferrin were analyzed.The results are summarized in Table 1. TABLE 1 Microinjected CellNon-Microinjected Cell Ave⁵⁾ Ave DNA¹⁾ Ab²⁾ Tf³⁾ Total⁴⁾ (%) Tf Total(%) PcDNA3 Anti- 1 12 7.50 4 32 11.09 rabbit 1 15 3 31 IgG Ab Ap-1⁶⁾Anti- 1 12 7.50 18 46 36.81 gene + rabbit 1 15 10 29 pcDNA3 IgG Ab Anti-1 12 10.42 18 37 48.65 amphi- 2 16 physin-1 Ab¹⁾microinjected DNA;²⁾antibody for analyzing microinjected cell;³⁾transferrin;⁴⁾total cell number;⁵⁾average value; and⁶⁾amphiphysin-1.

As known in Table 1, compared to cells microinjected with pcDNA3 asmock, the microinjected cells with pcDNA3 carrying amphiphysin-1 geneshows much higher transferrin-uptake activity. In other words, theendocytic activity of senescent cells was sharply increased by theintroduction of amphiphysin-1 cDNA. These results successfullydemonstrate that amphiphysin-1 is essential for the restoration offunctional endocytosis of the senescent cells and thus is essential formodulating cellular senescence.

EXAMPLE IX Cloning of Dominant Negative Amphiphysin-1 Gene

Dominant negative amphiphysin-1 gene, which is known to block thefunction of amphiphysin-1 (Shupliakov, O. et al., Science,276:259(1997); and Wigge P., Curr. Biol., 7:554(1997)), was amplified byPCT using amphiphysin-1 cDNA as template and specific primers, therebyamplifying a partial nucleotide sequence encoding the middle part ofamphiphysin-1 protein (amino acids 250 to 588). The forward primer andreverse primer used have the following sequences:5′-AACTGTCCACCATGAGTGATTCGGGTCCTCTCCGC-3′ and5′-GGATCCCTACTGCTCCGTAGCCAGCTCCGG-3′, respectively. The PCRamplification was performed and the amplified product was subclonedusing pcDNA 3 (Invitrogen) in the same manner as Example VII. Thegenetic map of the final vector is shown in FIG. 11.

EXAMPLE X Suppression of Endocytic Function in Young Cell by DominantNegative Amphiphysin-1 Gene

Using the vector constructed in Example IX, young fibroblasts (PDL 16)were transformed as follows: Two μg of the vector constructed in Example1× and 0.5 μg of pEGFP-N1 vector (Clontech) were mixed with DMEM and 8μl of Plus reagent, after which the resultant was allowed to stand for15 min. at room temperature. Thereafter, the mixture was mixed well withLipofectamine (Gibco-BRL) and DMEM, followed by standing the mixture for15 min. at room temperature. Following the further addition of 2 ml ofDMEM, the final mixture was added to young fibroblasts and thenincubated for 3 hrs at 37° C. After the lapse of 3 hr., 2.5 ml of DMEMcontaining 20% FBS were added and incubated for another 40 hr. Theincubated cells were treated with 25 μg/ml rhodamine-conjugatedtransferrin for 10 min. and the image was observed by confocalmicroscope (Biorad, #MRC1024), thereby elucidating the internalizationof transferrin either in transformed cell (emitting EGFP-derived greenfluorescence) or in non-transformed cell (see FIG. 12). As shown in FIG.12, co-transformed cells with the vector carrying dominant negativeamphiphysin-1 cDNA and pEGFP-N1 (left panel) show no red fluorescence byrhodamine-conjugated transferrin, indicating that the internalization ofrhodamine-conjugated tansferrin does not occur, but non-transformedcells (right panel) show red fluorescence.

These results demonstrate that the functional incompetence ofamphiphysin-1 can inhibit receptor-mediated endocytosis and finallyinduce cellular senescence.

EXAMPLE XI Analysis of Erk-1/2 Activation by Western Blotting

Young cells (PDL less than 30), middle cells (PDL 35-45) and old cells(PDL more than 60) of Human fibroblasts or IMR-90 cells, respectivelywere stimulated with 100 ng/ml EGF (Gibco-BRL, human, recombinant).After stimulation, western blotting was performed as described inExample V. Monoclonal anti-phospho-Erk-1/2 antibody, polyclonalanti-Erk-1/2 antibody and polyclonal anti-EGFR antibody were purchasedfrom Santa Cruz Biotechnology, Inc. As found in FIG. 13 a, in both youngand middle-aged cells, Erk-1/2 kinases were phosphorylated (activated)within 5 min, and the resulting activation was sustained for 15 minafter EGF stimulation. However, the phosphorylation of Erk-1/2 kinasefrom old cells was not detected until 15 min had lapsed. The expressionlevel of Erk kinase and and EGFR was not changed by the increase ofpopulation doubling in Western blot (see FIG. 13 b), despite thedown-regulation of EGF signaling to Erk kinases.

Therefore, the reduced responsiveness of old cells to growth factor isdue to the reduced Erk-1/2 phosphorylation, i.e., the reduced Erk-1/2activation.

EXAMPLE XII Analysis of Caveolin Expression by Western Blotting

Analysis of caveolin expression, in young cells (PDL less than 30),middle cells (PDL 35-45) and old cells (PDL more than 60) of Humanfibroblasts or IMR-90 cells, was performed by Western blotting asdescribed in Example V. Monoclonal anti-caveolin-1 antibody, monoclonalanti-caveolin-2 antibody and monoclonal anti-caveolin-3 antibody werepurchased from Transduction Laboratories. As shown in FIG. 14, withaging, all of caveolin-1, caveolin-2 and caveolin-3 were expressedincreasingly in both human fibroblasts and IMR-90 cells.

EXAMPLE XIII Analysis of Interaction between EGFR and Caveolin-1 byImmunoprecipitation

Young (PDL less than 30) or old (PDL more than 60) fibroblasts werelysed in IP lysis buffer (10 mM phosphate buffer, pH 7.4, 150 mM NaCl,1% Nonidet P-40, 2 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 2 μg/mlaprotinin, 2 μg/ml leupeptin, 50 mM NaF, 0.2 mM Na₃VO₄) and sonicatedbriefly. Lysates were spin down at 9,000 rpm for 5 min., andsupernatants were incubated with normal mouse serum, anti-EGFR antibodyor anti-caveolin-1 antibody. Immune complexes were precipitated withprotein A-Sepharose beads (Amersham Pharmacia Biotech) and separated bySDS-polyarylamide gel electrophoresis and analyzed by Western blot asdescribed in Example V (see FIG. 15).

As demonstrated in FIG. 15, the immune complex of EGFR from oldfibroblasts contained caveolin-1 proteins, while young fibroblasts didnot show a comparable amount of caveolin-1 and subsequent interactionswith EGFR.

It is elucidated that with aging, the expression of caveolin-1 proteinis increased and then the increased caveolin-1 protein is interactedwith EGFR to inhibit the activation of Erk-1/2 kinase by EGFR.

EXAMPLE XIV Electron Microscopic Analysis for Caveolin-1

Subconfluent young (PDL 20) and old (PDL 65) fibroblasts were palletizedby centrifugation (1,000 rpm) and fixed with 3%glutaraldehyde/phosphate-buffered saline at pH 7.4. After washing with0.2 M sodium cacodylate buffer, pH 7.4, cell pellets were treated with1% osmium tetroxide in cacodylate buffer for 1 hr. The cells were thendehydrated in graded ethanol steps through propylene oxide and embeddedin Embed812 (Electron Microscope Sciences). The embedded cells were cutto the size of 200 nm by microtomb (Leichert-JUNG) and the cuts werestained with methylene blue and azure II, followed by observation withlight microscopy in order to select the appropriate observation regionof electron microscopy. Thereafter, the selected region was ultra-cut tothe size of 60 nm by microtomb (Leichert-JUNG) and stained with uranylacetate and lead citrate. Sections were observed using a transmissionelectron microscope (H-600, Hitachi). FIG. 16 indicates that oldfibroblasts contain significantly more caveolae-like structure thanyoung cells.

EXAMPLE XV Construction of Plasmid DNA Carrying Caveolin-1 cDNA

Total RNA was isolated from human old fibroblasts using TRIzol(Gibco-BRL, #15596-026) and RT-PCR was then carried out to obtaincaveolin-1 cDNA using the isolated RNA. The primers used are: forwardprimer, 5′-atccaagcttccaccatgtctgggggcaaatacgt-3′ and reverse primer,5′-gcaggatccctatatttctttctgcaagttgat-3′. The reverse transcriptase andTaq polymerase used are AMT RTase from Promega and Ex Taq from TaKaRa,respectively. The temperature is set: 60° C. for 30 sec (annealing), 72°C. for 50 sec (extension) and 92° C. for 30 sec. (denaturation). Theamplified cDNA was verified by DNA sequencing in accordance with chaintermination method (Sanger, F. et al., Proc. Natl. Acad. Sci.,74:5463(1977)). The nucleotide sequence of caveolin-1 cDNA is found inSEQ ID NO:3. The amplified cDNA was subcloned into pcDNA3 (Invitrogen)using restriction sites of HindIII and BamHI. The genetic map of thefinal constructed vector is shown in FIG. 17.

EXAMPLE XVI Transformation of Human Fibroblasts with Caveolin-1 cDNA

Young human fibroblasts (PDL 25) were transformed using the vectorconstructed in Example XV. The cells were plated into the dish andincubated for 18 hrs to allow 70-80% confluency. Two μg of the vectorconstructed in Example XV was mixed with 8 μl of Plus reagent and theresulting mixture was mixed with 12 μl of Lipofectamine (Gibco-BRL) and238 μl of DMEM, followed by standing the mixture for 15 min. at roomtemperature. Following the further-addition of 2 ml of DMEM, the finalmixture was added to young fibroblasts and then incubated for 3 hrs at37° C. After the lapse of 3 hr., 2.5 ml of DMEM containing 20% FBS wereadded and incubated for another 24 hr. After. 30 hr. the transfectedcells were stimulated with 100 μg/ml EGF. Finally, Western blotting wascarried out as described in Example V so that the activation of Erk-1/2may be detected. In FIG. 18, lane 1 of panel A represents sampletransformed with pcDNA 3 and lane 2 represents sample transformed withpcDNA 3 carrying caveolin-1 cDNA. As verified in FIG. 18, the cellstransformed with pcDNA 3 carrying caveolin-1 cDNA express caveolin-1protein. In FIG. 18, lanes 1-3 of panel B represent samples transformedwith pcDNA 3 and subsequently stimulated with EGF for 0, 5 and 20 min,respectively, and lanes 4-6 of panel B represent samples transformedwith pcDNA 3 carrying caveolin-1 cDNA and subsequently stimulated withEGF for 0, 5 and 20 min.

As shown in FIG. 18, the expression of Erk-1/2 kinase was not changed incells overexpressing caveolin-1 protein whereas the phosphorylation ofErk-1/2 was significantly inhibited in comparison with mock-transformedcells.

These data demonstrate that the activation of Erk-1/2 kinase is blockedwhen introducing caveolin-1 DNA into cells and sequentially theresponsiveness to stimuli is diminished, thereby leading to cellularsenescence. The result reveals that the diminished activation of Erk-1/2kinase is due to the diminished expression level of caveolin-1 protein.

EXAMPLE XVII Transfection of Antisense Oligonucleotide to Caveolin-1

XVII-1: Synthesis of Antisense Oligonucleotide to Caveolin-1

To prepare antisense oligonucleotide to inhibit the expression ofcaveoin-1 protein, a suitable region of caveolin-1 mRNA to interact withantisense oligonucleotide was selected. Thus, the antisenseoligonucleotides capable of binding to translational initiation regionof caveolin-1 mRNA, which are designed to block translationalinitiation, were synthesized. The synthesized oligonucleoties wereconjugated with fluorescien (Genotech) in their 5′-terminal region andmodified by phosphorothioate to increase their stability. For example,the synthesized antisense oligonucleotide has a nucleotide sequence:5′-tttgcccccaga-3′. The sense oligonucleotide bound to the above regionwas also synthesized: 5′-atgtctgggggc-3′.

XVII-2: Transfection of Antisense Oligonucleotide to Caveolin-1

Old human fibroblasts (PDL 64) were placed onto 24 well-plate or dishcontaining DMEM without FBS and incubated in incubator (37° C., 5% CO₂)for 12 hr. After incubation, 1-5 μg of 100 μM antisense oligonucleotidessynthesized and Plus reagent (Gibco-BRL) were mixed with 500 μl of DMEMand subsequently reacted for 15 min. at room temperature, and theresulting mixture was well mixed with 500 μl of the mixture containing12 μl of Lipofectamine (Gibco-BRL) and DMEM, followed by standing themixture for 15 min. at room temperature in order to form liposomecomplex. The incubated cells were washed twice with DMEM (no serum) andtreated with 1 ml of the liposome complex, followed by incubation for 3hr at 37° C. To the transfected cells, 1-5 ml of DMEM containing 10% FBSwas added and the incubation was subsequently carried out for 24 hr,after which the media was changed with DMEM containing 10% FBS.Following incubation for a given period, immunostaining and westernblotting were carried out as follows:

XVII-3: Immunostaining

The cells treated with antisense oligonucleotide were fixed with 0.5 mlof 4% paraformaldehyde for 20 min at room temperature and thenpermeabilized with 0.5 ml of 0.5% Triton X-100 in PBS for 10 min,followed by blocking with 2% BSA (in PBS). The cells were sequentiallyincubated with anti-caveolin-1 antibody (Transduction Laboratory)overnight at 4° C. and then rhodamine-conjugated secondary antibody(Santa Cruz) for 1 hr at room temperature. For the purpose ofvisualizing nucleus, DAPI (Molecular Probe) was also added. Theobservation was performed using confocal microscope (Biorad, #MRC1024).As shown in FIG. 19, the expression of caveolin-1 in cell isdramatically decreased in the cells treated with antisenseoligonucleotide with a lapse of treatment time, while the expression ofcaveolin-1 is not changed in the cells treated with senseoligonucleotide. Interestingly, the old cells treated with antisenseoligonucleotide exhibited the altered cell morphology: enlarged andspread morphology to smaller and spindle morphology. In contrast, theold cells treated with sense oligonucleotide did not show suchcell-morphology alteration.

XVII-4: Western Blotting

Western blotting was performed with the cells treated witholigonucleotides in the same manner as Example XII. The cells treatedwith antisense oligonucleotide provided a weaker band corresponding tocaveolin-1, indicating that the expression of caveolin-1 is decreased inthe cells treated with antisense oligonucleotide.

Based on the results from immunostaining and western blotting, it iselucidated that caveolin-1 is directly involved in cellular senescenceand the inhibition of expression of caveolin-1 leads to not only theprevention of cellular senescence but also the conversion of old cell toyoung cell.

EXAMPLE XVIII Analysis of Erk-1/2 Activation Upon Transfection ofAntisense Oligonucleotide to Caveolin-1

The old and young fibroblasts were treated with antisenseoligonucleotide as described in Example XVII. The EGF stimulation andwestern blotting were carried out as described in Example XI. FIG. 20represents the results of this Example. As shown in FIG. 20, Erk-1/2kinases in young cells were strongly phosphorylated (activated).However, non-treated old cells and treated old cells with senseoligonucleotide showed higher basal Erk-1/2 activity than young cellsand when stimulated with EGF, the cells showed no alteration in Erk-1/2activation. Interestingly, in old cells treated with antisenseoligonucleotide, Erk-1/2 activation by EGF was highly increased as youngcells.

These observations elucidates that the inhibition of caveolin-1expression due to the treatment with antisense oligonucleotide, provideold cells with the restoration of signal cascade mediated by Erk, whichis typical in young cells.

EXAMPLE XIX Observation of p-Erk-1/2 Translocation to Nucleus uponTransfection of Antisense Oligonucleotide to Caveolin-1

To verify that Erk-1/2 kinase activated in Example XVIII is translocatedinto nucleus and regulate sequentially the expression of other genes,immunostaining was performed as follows: Young and old fibroblasts weretreated with antisense oligonucleotide and EGF as Example XIII. Thetreated cells were sequentially incubated with anti-p-Erk(phosphorylated-Erk) antibody (New England Biotech) overnight at 4° C.and then FITC-conjugated secondary antibody (Santa Cruz) for 1 hr atroom temperature. For the purpose of visualizing nucleus, DAPI(Molecular Probe) was also added. The image of p-Erk-1/2 localizationwas visualized using confocal microscope (Biorad, #MRC1024), which isfound in FIGS. 21 and 22. In FIGS. 21 and 22, arrows indicatetranslocation of p-Erk-1/2 kinase into nucleus.

As shown in FIG. 21, in young cells, at 5 min after treatment, p-Erk-1/2was strongly observed in cytoplasm, at 30 min after treatment, p-Erk-1/2translocated into nucleus was seen and at 60 min after treatment,p-Erk-1/2 was weakly observed only in cytoplasm. These results indicatethat Erk-1/2 is activated (phosphorylated) within min after treatment,the resulted p-Erk-1/2 is translocated into nucleus to regulatetranscription of several genes at 30 min and is finally inactivated at60 min. In contrary to the young cells, old cells exhibited thatp-Erk-1/2 was strongly observed in cytoplasm irrespective of EGFtreatment, which is also found in the results of western blotting.Interestingly, old cells showed no p-Erk-1/2 translocated into nucleus.

Moreover, as shown in FIG. 22, old cells treated with senseoligonucleotide showed no p-Erk-1/2 in nucleus and vice versa for oldcells treated with antisense oligonucleotide.

These results demonstrate that the inhibition of the expression ofcaveolin-1 with antisense oligonucleotide is responsible for therestoration of Erk-mediated signal cascade. Furthermore, the resultsindicate that caveolin-1 is also involved in the restoration oftranslocation into nucleus, which is generally blocked in old cells.

EXAMPLE XX Methylation of CpG Island of Caveolin-1 Gene

It is well known that upon aging, the expression of p16/Ink4a isincreased with the decrease of the methylation level of CpG islandlocated in promoter thereof (Jarrard D F., Cancer Res.,15;59(12):2957-2964(1999)). Furthermore, it has been revealed thatcaveolin-1 also has a similar methylation pattern in cancer cell (CuiJ., Prostate, 15;46(3):249-256(2001)). Therefore, the inventors examinedwhether the decreased expression of caveolin-1 is ascribed to suchmethylation.

Young fibroblasts (PDL 20) were treated with 1 μM demethylating agent,5-aza-deoxycytidine (Sigma) in DMSO and then periodically treated withthe agent at the time of changing media for 2-3 weeks. The induction ofcellular senescence was verified by SA β-gal activity staining asExample III. To investigate the expression of related proteins, thetreated cells were harvested in different days and western blotting wasperformed as previously described. In western blotting, anti-p53antibody, anti-p16 antibody, anti-caveolin-1 antibody and anti-actinantibody were purchased from Santa Cruz Biotechnology, Inc.

As shown in FIG. 23 a representing the results of SA β-gal activitystaining, the young cells treated for about 2 weeks showed senescentcell-like phenomenon. As shown in FIG. 23 b representing the results ofwestern blotting, with demethylation, the expression of p16 andcaveolin-1 were increased and the expression of p53 was not altered.Interestingly, the increased level of p16 was detected at the earlyphase of cellular senescence, whereas the increased level of caveolin-1was detected earlier than p16. These results elucidate that theincreased level of caveolin-1 in senescent cells was not leaded directlyby either cellular senescence or the increased level of p16 but bydemethylation of CpG island in promoter of caveolin-1 gene.

As a result, it is revealed that cellular senescence can be modulated bythe methylation level of the promoter, in particular, CpG island ofcaveolin-1 gene. Having described a preferred embodiment of the presentinvention, it is to be understood that variants and modificationsthereof falling within the spirit of the invention may become apparentto those skilled in this art, and the scope of this invention is to bedetermined by appended claims and their equivalents.

1-24. (canceled)
 25. A method for modulating cellular senescence invitro, comprising introducing into a senescent cell a polynucleotideencoding an amphiphysin-1 protein involved in cellular senescence.26-29. (canceled)
 30. The method according to claim 25, wherein thepolynucleotide is gDNA or cDNA.
 31. The method according to claim 25,wherein the polynucleotide encoding the amphiphysin-1 protein involvedin cellular senescence is contained in an expression vector foreucaryotic cells. 32-48. (canceled)